Essentiality of the early transcript in the replication origin of the lactococcal prolate phage c2

Abstract

The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter P(E)1 abolished origin function. Introduction of mutations into P(E)1 which did not eliminate promoter activity or replacement of P(E)1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of P(E)1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the P(E)1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3' end of the DNA sequence coding for the transcript. The P(E)1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of P(E)1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

FIG. 1.

Schematic representation of the c2 ori locus. The positions of the origin fragment in the phage c2 genome relative to the early gene promoter (solid arrows) and the late gene promoter (open arrow) are shown at the top. Below this the c2 ori (positions 6717 to 7238 in the c2 sequence) and the derivatives of the origin fragment that were cloned in pVA891 are shown. The abilities of the various constructs (listed on the left) to replicate in L. lactis are indicated on the right as mean transformation frequencies from at least three experiments (in the case of pLP212 in the presence and in the absence of nisin); the values varied because of variations in the L. lactis transformation frequency between experiments. (A) The solid triangles indicate the position of the base pair changes in the pLP205 insert. The gray arrow represents the nisA promoter in the pLP212 insert. In pLP213 P_E1 is inverted. (B) Schematic representation of c2 ori fragments cloned in pVA891 carrying shortened DNA sequences coding for the transcript made from P_E1 (pLP201 to pLP211). The length of the remaining sequence of each transcript from the transcription start site is indicated above the transcript. The gray line represents the lactococcal prtP sequence replacing the c2 transcript-encoding sequence in the pLP214 insert. Ω in pLP216 represents the transcriptional and translational terminator cloned at the 3′ end of the 207-bp transcript-encoding sequence.

FIG. 2.

Schematic representation of plasmids used in scanning mutagenesis. The c2 ori fragments carrying deletions in the transcript-encoding sequence were cloned into pVA891 and assayed for the ability to replicate in L. lactis. The numbers above the lines indicate the sizes (in base pairs) of the fragments 5′ and 3′ of the deletion. The spaces between the solid lines indicate the positions of deletions, and the numbers preceded by Δ indicate the lengths of the deletions (in base pairs).

FIG. 3.

Primer extension analysis to identify the transcription start site in the pVA891-ori plasmids. The primer extension reaction was performed with total RNA isolated from L. lactis containing plasmid pLP201 or pLP203 by using primer 6902prex. The primer extension products in both lanes are indicated by the arrow. The same primer was used for the sequencing reaction with pLP201 (lanes A, C, G, and T). Sizes (in nucleotides) are indicated on the left.

FIG. 4.

Northern hybridization analysis of the c2 ori fragments that support plasmid replication. Twenty micrograms of total RNA from L. lactis cells harboring a plasmid was electrophoresed on an acrylamide gel, blot transferred, and then probed with a PCR product (template pLP206; primers ori1 and ori7) and detected by the ECL system. MG1363, no plasmid; pLP constructs, MG1363 transformed with the plasmids indicated; NZ9000 and NZ9800, two lactococcal host strains for pLP212, in which the ori transcript is placed under control of the nisA promoter; + nisin, growth in the presence of nisaplin; − nisin, growth in the absence of nisaplin.

FIG. 5.

Northern hybridization analysis of the c2 ori fragments that did not support plasmid replication. The fragments were recloned into a plasmid (pFX3) that contains an origin of replication for gram-positive bacteria. Twenty-microgram portions of total RNA from L. lactis strain MG1363 harboring the plasmids were electrophoresed on acrylamide gels, blot transferred, and then probed with different PCR products and detected by the ECL system. The migration positions of two marker bands (280 and 155 nt) are indicated on the left. Each lane is labeled with the designation of the plasmid construct (see Fig. 1 and 2 for schematic representations of the constructs). The following PCR-generated probes were used: lane pFX3-214, template pLP214 and primers ori11 and ori12; lane pFX3-213, template pFX3-213 and primers ori4 and M13 Rev; lanes pFX3-215, pFX3-223, pFX3-218, pFX3-219, pFX3-220, pFX3-221, pFX3-222, pFX3, pFX3-203, pFX3-205, pFX3-206, pFX3-207, pFX3-216, pFX3-208, and pFX3-204, template pLP206 and primers ori1 and ori7.